Iron-oxidizing microorganisms: genes, mechanisms, and environmental impact
Date
2025
Authors
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Publisher
University of Delaware
Abstract
Iron is the fourth most abundant element in Earth’s crust and plays a crucial role in the planet’s biogeochemical cycles. Freshwater iron-oxidizing bacteria (FeOB) can catalyze and accelerate iron oxidation thereby mediating, and potentially driving, iron cycling in diverse environments. However, without functional marker genes of iron oxidation, we are unable to fully assess FeOB contributions to environmental iron oxidation. This dissertation addresses that knowledge gap by identifying novel genes and mechanisms with a potential role in Fe oxidation, testing the expression of novel genes and known Fe oxidases in a lab isolate, and investigating the expression of those genes in the environment. It also explores the environmental impact of FeOB, examining how their Fe-cycling metabolisms can impact C and N cycling in wetland environments. ☐ We need a better accounting of iron oxidation genes and mechanisms before we can detect and quantify biotic iron oxidation in the environment. This is especially true for iron oxidation in freshwater environments, for which there are few isolates. Of the isolates we do have, many are difficult to culture, growing slowly and to low cell density, limiting our ability to investigate them. In the first study, we leveraged bioinformatics tools to do a pangenome analysis of the Gallionellaceae, a family of freshwater bacteria known for its iron-oxidizing members. We first resolved the Gallionellaceae phylogeny, identifying four main genera. We then compared genomes of the three iron-oxidizing genera (Gallionella, Sideroxydans, and Ferriphaselus) against genomes from a single nitrite oxidizing genus (Candidatus Nitrotoga) to learn about the genes and mechanisms unique to iron oxidation. Cyc2 and MtoA iron oxidase genes were widely distributed, yet specific to the FeOB. Further analysis of the FeOB genomes identified additional shared metabolic traits and genes that may encode adaptations for Fe oxidation and extracellular electron uptake (EEU). This includes genes that may encode iron oxidation pathway components (ircABCD), and genes for multiheme c-type cytochromes (MHCs) that could form porin-cytochrome complexes to oxidize Fe or transport electrons throughout a range of redox potentials. Overall, the Gallionellaceae pangenome provides an in-depth look at the metabolic flexibility and genetic potential of a family of FeOB. It suggests Gallionella, Sideroxydans, and Ferriphaselus have a range of adaptations that contribute to their success. It also identifies putative genes and mechanisms of Fe oxidation for additional study. ☐ Determining whether a gene’s expression is specific to a single activity is a key step in evaluating its potential as a functional marker gene. However, evidence of gene expression in FeOB remains limited, partly due to the challenges of culturing sufficient biomass and iron’s interference with DNA and RNA extraction and sequencing. In this study, we analyzed the gene expression of Sideroxydans sp. CL21 to investigate its iron oxidation genes and pathways. CL21 is a Gallionellaceae FeOB capable of growth on both organic and inorganic substrates, making it the only organotrophic Gallionellaceae FeOB isolate to date. It contains genes for both known iron oxidases (cyc2, mtoA) and several additional genes of interest identified in the Gallionellaceae pangenome (ircABCD, PCC3). To overcome the challenges of low biomass and iron interference, we grew CL21 on lactate instead of Fe(II) and used iron oxidation assays to determine when cells were able to oxidize iron. These iron assays revealed lactate-grown cells could oxidize Fe(II) during late-log phase (Day 14), but not at mid-log phase (Day 6). This allowed us to compare gene expression between iron-oxidizing (late-log) and non-iron-oxidizing (mid-log) cells. We found iron oxidase genes (cyc2, mtoA) were highly expressed at both time points, and did not correspond to iron oxidation activity. This supports growing evidence that cyc2 expression is often high and is not (by itself) a sign of iron oxidation activity. In contrast, several genes encoding periplasmic and inner membrane cytochromes (mtoD, cymA/imoA, ircABCD) were significantly upregulated at the late-log (iron-oxidizing) timepoint. These expression patterns suggest CL21’s iron oxidation pathway may be regulated at different stages, with genes encoding periplasmic and inner membrane electron carriers (mtoD, cymA/imoA, ircABCD) upregulated at times of iron oxidation activity. Thus, genes like ircABCD may be prospective indicators of iron oxidation activity in the environment. ☐ Minerotrophic wetlands (fens) are home to diverse microbial communities that drive biogeochemical cycling, but the understanding of Fe-cycling organisms in these environments is limited. In particular, the taxonomic and metabolic diversity of these iron-cycling communities is not well understood. In this third study, we used our insights into the genes and mechanisms of Fe oxidation along with knowledge of Fe reduction and N-cycling genes to assess the microbial community of the Schlöppnerbrunnen fen. We found that nearly half the reconstructed community had a potential for Fe-cycling at all depths (5-30 cm). Fe oxidizers included many Gallionellaceae from known (Gallionella, Sideroxydans) and novel (RI-121, CAITJF01) genera. Surprisingly, an abundant and active Bathyarchaeia (FEN-987) also encoded and expressed a Cluster 1 cyc2. Fen FeRB were also diverse. Known taxa, like Geobacter, made up a small percent of the community while members of the Acidobacteriae with iron reduction genes appeared more abundant. By analyzing these FeOB and FeRB in conjunction with N-cycling, we found multiple organisms with a potential for NDFO or Feammox in the fen. This suggests iron cyclers may be able to directly influence N transformations. Overall, this study identified a diverse microbial community with a high potential for Fe-cycling. It shows that not only do wetlands have a high capacity for biogeochemical Fe- and N-cycling, but those processes may be linked though NDFO or Feammox metabolisms in various organisms making the continued study of Fe-cyclers key to understanding biotic drivers of N detoxification and GHG production. ☐ Overall, this dissertation contributes to the development of functional gene markers for Fe oxidation by leveraging -omics and bioinformatics analyses of both lab isolates and environmental datasets. It identifies key genes and mechanisms for further investigation, while also advancing our understanding of the environmental role of FeOB and their involvement in coupling the Fe, C, and N cycles.
Description
Keywords
Gallionellaceae, Iron oxidation, Iron-oxidizing bacteria, Sideroxydans, Wetland iron-cycling